Gap junction channels, dodecamers of connexin proteins, provide direct coupling between the cytoplasm of adjacent cells, each cell contributing a hexameric connexon, or hemichannel. Mutations in connexin genes are associated with diseases including X-linked Charcot-Marie-Tooth disease (CMTX), a peripheral neuropathy caused by defects in connexin32. The applicant aims to characterize the pharmacology of conducting hemichannels formed by human connexin37 (hCx37) and bovine connexin44 (Cx44), which have diverse properties with respect to gating. Experiments are proposed to test the hypothesis that block of these hemichannels by heptanol, halothane, and divalent cations should be the same in hemichannels as in intact intercellular channels, indicating that gap junctional uncoupling is due to hemichannel block. Voltage effects on divalent block will also be assessed. Single channel currents for hCx37 will determine whether the hemichannel currents have a role under physiological conditions. Native preparations will be used to further explore this possibility. Finally, chimeric channels, containing domains of the hCx37 and U44, will be made to determine the molecular basis of gating. Chemical gating properties will be assessed in chimeric hemichannels that exchange the cytoplasmic loop and carboxy-terminal domains, regions implicated in chemical gating of connexins. Voltage gating properties will be examined in hemichannels and intercellular channels formed by chimeras that exchange the extracellular loop domains. Site-directed mutants can also be made in the domains involved in gating. Once the molecular basis of gating is identified, specific residues may be used as targets for rationale drug design to treat illnesses like CMTX.